An Improved Protocol for Isolation and Culturing of Mouse Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs) represent a unique testicular cell type that has the capacity for proliferating, differentiating, and transmitting genetic information. This particular cell type is a strong focus of stem cell research, with isolation and maintenance of SSCs as an important issue. Therefore, we attempted to optimize SSCs handling and to analyze different media and feeder layers, such as adult and embryonic Sertoli cells. The expression patterns of SSC-specific proteins (α6 and β1 integrins, Stra8, and DAZL) and restoration of spermatogenesis were chosen as parameters to demonstrate the efficacy of the protocol. SSCs were isolated from testes of 3- to 6-day-old mice using a magnetic activated cell-sorting system and Thy-1 antibody. After enrichment, SSCs were cultured for 7 days with different media and feeder layers. Then, SSCs were transplanted to recipient mice. Culturing on adult and embryonic Sertoli cells and in the presence of different growth factors [glial cell line-derived neurotrophic factor (GDNF), GDNF family receptor α1 (GFR-α1), and basic fibroblast growth factor (bFGF) resulted in an undifferentiated SSC phenotype with typical stem cell characteristics observed in vivo. The established co-culture model could help to improve the recovery and quality of stem cell preparation of mammalian testis.